Abstract
The kinetic and thermodynamic consequences of intrinsic disorder in protein-protein recognition are controversial. We address this by inducing one partner of the high-affinity colicin E3 rRNase domain-Im3 complex (K(d) ≈ 10(-12) M) to become an intrinsically disordered protein (IDP). Through a variety of biophysical measurements, we show that a single alanine mutation at Tyr507 within the hydrophobic core of the isolated colicin E3 rRNase domain causes the enzyme to become an IDP (E3 rRNase(IDP)). E3 rRNase(IDP) binds stoichiometrically to Im3 and forms a structure that is essentially identical to the wild-type complex. However, binding of E3 rRNase(IDP) to Im3 is 4 orders of magnitude weaker than that of the folded rRNase, with thermodynamic parameters reflecting the disorder-to-order transition on forming the complex. Critically, pre-steady-state kinetic analysis of the E3 rRNase(IDP)-Im3 complex demonstrates that the decrease in affinity is mostly accounted for by a drop in the electrostatically steered association rate. Our study shows that, notwithstanding the advantages intrinsic disorder brings to biological systems, this can come at severe kinetic and thermodynamic cost.
Highlights
intrinsically disordered protein (IDP) have been retained in biological systems over evolutionary time, this implies that they endow these systems with particular advantages over their globular counterparts.3b One of the often cited advantages of IDPs in protein−protein interaction (PPI) is their faster association rate, due to a larger capture radius, the so-called “fly-casting” mechanism, and fewer encounters on the path to the final complex.[5]
The 12-kDa E3 ribosomal RNase (rRNase) domain is delivered to the cytoplasm of susceptible bacteria, where it cleaves the phosphodiester bond between A1493 and G1494 within the decoding center of the ribosomal A-site, leading to the inhibition of protein synthesis and cell death.[6]
We determined the hydrodynamic radius of E3 rRNaseIDP by NMR spectroscopy (25.6 Å) and found it midway between those of the native domain (20.1 Å) and E3 rRNase unfolded in 8 M urea (30.2 Å), suggesting that, like other IDPs,[11] E3 rRNaseIDP is more compact than the urea-denatured state
Summary
IDPs have been retained in biological systems over evolutionary time, this implies that they endow these systems with particular advantages over their globular counterparts.3b One of the often cited advantages of IDPs in PPIs is their faster association rate, due to a larger capture radius, the so-called “fly-casting” mechanism, and fewer encounters on the path to the final complex.[5]. Demonstrated that the thermodynamic consequence of inducing the E3 rRNase to become an IDP (at pH 7.0 and 25 °C) was a 4 orders of magnitude increase in the equilibrium dissociation constant with respect to that of the WT complex: Kd = 28 nM compared to 1.2 pM at 25 °C, pH 7.0, 200 mM NaCl (Figure S2D and Table 1).
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